Children with genetic diseases involving the lung including cystic fibrosis may require lung transplantation. However patients who experience an increased number of acute rejection episodes are at risk for the development of chronic rejection (obliterative bronchiolitis [OB]). This results in a 5-year survival of less than 50%. New strategies to prevent acute and chronic rejection after lung transplantation are required. The establishment of mixed hematopoietic chimerism (MC) requires 2-way tolerance (graft vs. host and host vs. graft) to hematopoietic tissue and is associated with the development of organ-specific tolerance. Lung allografts have been successfully transplanted from the hematopoietic cell (HC) donor to recipients with stable MC without the need for immunosuppression. These recipients have been followed long-term without the development of acute or chronic rejection. This model of HC and lung graft tolerance has been further characterized and the findings are consistent with a peripheral mechanism involving regulatory (CD4+ CD25+) T cells. Recipient-derived donor-specific alloreactive T cells can be identified in vitro in blood from mixed hematopoietic chimeras with a donor-derived solid organ graft after removal of CD4+ CD25+ regulatory T cells. Therefore in this model of immune tolerance, deletional mechanisms have not been observed to make a significant contribution. This is in contrast to rodent models of MC in which deletional mechanisms have been observed and presumed to have a considerable role in the establishment of tolerance. Regulatory mechanisms of tolerance may also be relevant for transplantation of lung grafts in the absence of a hematopoietic stem cell graft. Therefore the goal of this grant proposal is to further investigate the role of regulatory T cells in the induction of immune tolerance to lung allografts in a large animal model (dog). We have established the methods for ex vivo expansion of regulatory T cells and can produce sufficient quantities to conduct in vivo studies. We will investigate if addback of ex vivo expanded recipient derived CD4+CD25+ regulatory T cells without HCT is safe and will achieve the goal of prolonged lung graft survival in young recipients (4-6 months old). Cell dosing and scheduling strategies for addback of regulatory T cells in combination with lung transplantation will be assessed. Fate of the ex vivo expanded regulatory cells after infusion will also be characterized. This will contribute to the understanding of the role these cells have in the establishment of tolerance. Development of preclinical strategies for the induction of lung-specific tolerance in large animal models to prevent acute and chronic rejection will increase the probability of successful translation to human studies. [unreadable] [unreadable] [unreadable]